This invention relates to a new family of receptors, which includes the growth hormone secretagogue receptors (GHSRs) and growth hormone secretagogue-related receptors (GHSRRs), nucleic acids encoding these receptors; and to the use of a GHSR to identify growth hormone secretagogues and compounds that modulate GHSR function.
Growth hormone (GH) is an anabolic hormone capable of promoting linear growth, weight gain and whole body nitrogen retention. Classically, GH is thought to be released primarily from the somatotroph cells of the anterior pituitary under the coordinate regulation of two hypothalamic hormones, growth hormone releasing factor (GHRF or GRF) and somatostatin. Both GHRF stimulation and somatostatin inhibition of the release of GH occurs by the specific engagement of receptors on the cell membrane of the somatotroph.
Recent evidence has been mounting which suggests that GH release is also stimulated by a group of short peptides, the growth hormone releasing peptides (GHRP; GHRP-6, GHRP-2 [hexarelin]) which are described, for example, in U.S. Pat. No. 4,411,890, PCT Patent Pub. No. WO 89/07110, PCT Patent Pub. No. WO 89/071 11, PCT Patent Pub. No. WO 93/04081, and J. Endocrinol Invest., 15 (Suppl 4), 45 (1992). These peptides function by selectively binding to distinct somatotroph cell membrane receptor, the growth hormone secretagogue receptor(s) (GHSRs). A medicinal chemical approach has resulted in the design of several classes of orally-active, low molecular weight, non-peptidyl compounds which bind specifically to this receptor and result in the pulsatile release of GH. Such compounds possessing growth hormone secretagogue activity are disclosed, for example, in the following: U.S. Pat. Nos. 3,239,345; 4,036,979; 4,411,890; 5,206,235; 5,283,241; 5,284,841; 5,310,737; 5,317,017; 5,374,721; 5,430,144; 5,434,261; 5,438,136; 5,494,919; 5,494,920; 5,492,916; EPO Patent Pub. No. 0,144,230; EPO Patent Pub. No. 0,513,974; PCT Patent Pub. No. WO 94/07486; PCT Patent Pub. No. WO 94/08583; PCT Patent Pub. No. WO 94/11012; PCT Patent Pub. No. WO 94/13696; PCT Patent Pub. No. WO 94/19367; PCT Patent Pub. No. WO 95/03289; PCT Patent Pub. No. WO 95/03290; PCT Patent Pub. No. WO 95/09633; PCT Patent Pub. No. WO 95/11029; PCT Patent Pub. No. WO 95/12598; PCT Patent Pub. No. WO 95/13069; PCT Patent Pub. No. WO 95114666; PCT Patent Pub. No. WO 95/16675; PCT Patent Pub. No. WO 95/16692; PCT Patent Pub. No. WO 95/17422; PCT Patent Pub. No. WO 95/17423; PCT Patent Pub. No. WO 95/3431 1; PCT Patent Pub. No. WO 96/02530; Science, 260, 1640-1643 (Jun. 11, 1993); Ann. Rep. Med. Chem., 28, 177-186 (1993); Bioorg. Med. Chem. Ltrs., 4(22), 2709-2714 (1994); and Proc. Natl. Acad. Sci. USA 92, 7001-7005 (July 1995).
The use of orally-active agents which stimulate the pulsatile release of GH would be a significant advance in the treatment of growth hormone deficiency in children and adults as well as provide substantial benefit under circumstances where the anabolic effects of GH might be exploited clinically (e.g. post-hip fracture rehabilitation, the frail elderly and in post-operative recovery patients).
It would also be desirable to know the molecular structure of growth hormone secretagogue receptors in order to analyze this new receptor family and understand its normal physiological role in concert with the actions of GHRF and somatostatin. This could lead to a better understanding of the in vivo processes which occur upon ligand-receptor binding. Further, it would be desirable to use cloned-growth hormone secretagogue receptors as essential components of an assay system which can identify new growth hormone secretagogues.
This invention relates to a novel family of receptors which includes growth hormone secretagogue receptors (GHSRs) and growth hormone secretagogue-related receptors (GHSRRs).
A first aspect of this invention are the growth hormone secretagogue receptors, which are free from receptor associated proteins. GHSRs may be from any species, and in further embodiments may be isolated or purified. One embodiment of this invention is human growth hormone secretagogue receptor (hGHSR), free from receptor-associated proteins. A further aspect of this invention is hGHSR which is isolated or purified.
Another aspect of this invention is swine growth hormone secretagogue receptor (sGHSR), free from receptor-associated proteins. A further aspect of this invention is sGHSR which is isolated or purified.
Another aspect of this invention is rat growth hormone secretagogue receptor (rGHSR), free from receptor-associated proteins. A further aspect of this invention is sGHSR which is isolated or purified.
Another aspect of this invention are human, swine and rat GHSRs which are encoded by substantially the same nucleic acid sequences, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native forms. These variant forms may have different and/or additional functions in human and animal physiology or in vitro in cell based assays.
Another aspect of this invention are the growth hormone secretagogue-related receptors, free from associated receptor proteins. A further embodiment are GHSRRs which are isolated or purified. These may be from any species, including human, mouse, rat and swine.
Growth hormone secretagogue receptors are proteins containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. In accordance with this invention, it has been shown that the N-terminal portions of the GHSR are not essential for its activation by the Growth Hormone Secretagogues (GHSs). Thus this invention specifically includes modified functionally equivalent GHSRs which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent GHSRs which contain modified and/or deletions in other domains, which are not accompanied by a loss of functional activity.
Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.
A further aspect of this invention are nucleic acids which encode a growth hormone secretagogue receptor or a functional equivalent from swine, human, rat or other species. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode a GHSR or a functional equivalent.
Yet another aspect of this invention relates to vectors which comprise nucleic acids encoding a GHSR or a functional equivalent. These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode a GHSR. It is well within the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
A further aspect of this invention are host cells which are transformed with a gene which encodes a growth hormone secretagogue receptor or a functional equivalent. The host cell may or may not naturally express a GHSR on the cell membrane. Preferably, once transformed, the host cells are able to express the growth hormone secretagogue receptor or a functional equivalent on the cell membrane. Depending on the host cell, it may be desirable to adapt the DNA so that particular codons are used in order to optimize expression. Such adaptations are known in the art, and these nucleic acids are also included within the scope of this invention. Generally, mammalian cell lines, such as COS, HEK-293, CHO, HeLa, NS/O, CV-1, GC, GH3 or VERO cells are preferred host cells, but other cells and cell lines such as Xenopus oocytes or insect cells, may also be used.
Growth hormone secretagogue related receptors are related to GHRS, but are encoded by a distinct gene. The GHRR genes may be identified by hybridization (using relaxed or moderate stringency post-hybridizational washing conditions) of cDNA of GHR DNA to genonic DNA. These sequences have a high degree of similarity to GHR.
Another aspect of this invention is a process for identifying nucleic acids encoding growth hormone secretagogue related receptors comprising hybridizing a first nucleic acid encoding a growth hormone secretagogue receptor with a second nucleic acid suspected of comprising nucleic acids encoding a growth hormone secretagogue, wherein the hybridizing takes place under relaxed or moderate post hybridizational washing conditions; and identify areas of the second nucleic acid where hybridization occurred.